The yeast, Saccharomyces cerevisiae, has multiple plasma membrane permeases that transport amino acids into the cell. These amino acids can either be metabolized and used by the cell as a principal nitrogen source for growth or be utilized directly in protein synthesis. The control of amino acid uptake is complex and not well understood. Study of particular transport systems has been complicated by the redundancy of the process, most permeases can transport several substrates with varying efficiency and most amino acids can be imported by more than one permease. Loss of an individual permease has little, or no, effect on cells growing in lab conditions and thus the 'wild-type' strains in general lab use are highly variable in permease content. This also complicates studies of transport in different strain backgrounds. The known permease genes which have been cloned and sequenced show considerable homology with certain absolutely conserved features. Thus, as the Yeast Genome Project proceeds to identify new genes, more 'putative' amino acid permeases are being identified. In this project, the substrate range and specificity of these unknown permeases will be elucidated by systematic deletion of the permease genes followed by careful analysis of changes in the amino acid uptake add sensitivity to amino acid analogs of the resulting deletion strains. When all the permeases responsible for transport of a particular amino acid have been characterized a detailed kinetic analysis of transport can be completed. Also, strains in which the permease content is assured can be constructed which will be made available to the yeast community. In a separate set of experiments, the function of the highly conserved regions of the permease molecules will be investigated. It is likely that these regions will either be important in amino acid transport and regulation or in localization of the permease through the secretory pathway to the plasma membrane. These possibilities will be systematically tested by directed mutagenesis of these regions in two representative permeases.